Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.Bladder cancer occurs in multiple forms, the most common of which is urothelial carcinoma (UCC), which represents >90% of all bladder cancers.¹ Approximately 30 to 50% of patients with invasive bladder cancer into the muscular wall of the bladder will develop metastatic disease and die within 2 years of diagnosis.² In addition, virtually all patients diagnosed with distant UCC metastases will succumb to disease.³ Currently, the standard treatment modality for muscle-invasive bladder cancer is radical cystectomy; systemic chemotherapy is generally reserved for patients with metastatic disease, although these treatment regimens provide only a limited long-term benefit with only rare reports of complete remission.^4,5 In light of these clinical outcomes, identification of new therapeutic targets is needed to define potential additional treatment avenues for these patients.Activation of the mammalian target of rapamycin (mTOR) signaling pathway occurs in many cancers and has recently been shown to correlate with more aggressive disease behavior,^6,7,8,9 although it has not been examined in great detail in UCC. Activation of mTOR occurs via a multistep process that includes upstream phosphoinositide-3 kinase (PI3K) and AKT activation, leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) heterodimer.^10,11 Inactivation of this heterodimer results in release of Rheb inhibition and subsequent mTOR activation by means of Rheb GTPase activity. Once activated, mTOR can induce increased mRNA translation or regulate the actin cytoskeleton via differential association Rictor and Raptor proteins.^10,11 Ultimately, mTOR activity regulates the effects of a number of downstream molecules important in cellular growth, including p70 S6 kinase-1 (S6K) and elongation-initiation factor 4E binding protein-1 (4E-BP1). Selective inhibition of the mTOR pathway can be achieved using rapamycin or rapamycin analogs temsirolimus (CCI-779, Wyeth Pharmaceuticals) and everolimus (RAD001, Novartis), which are currently in use in numerous clinical trials for solid tumors, with promising results in patients with advanced renal cell carcinoma.^12,13To further investigate the potential role of mTOR signaling and inhibition in UCC of the bladder, we used human cancer specimens, xenograft models, and in vitro analysis to determine the effects of mTOR on cellular proliferation, apoptosis, tumor growth, and clinical outcomes in this cancer population.